The human astrovirus RNA-dependent RNA polymerase coding region is expressed by ribosomal frameshifting

Abstract

The genomic RNA of human astrovirus serotype 1 (HAst-l) contains three open reading frames (ORFs), la, lb, and 2. ORF lb is located downstream of, and overlaps, la, and it has been suggested on the basis of sequence analysis that expression of ORF lb is mediated through-1 ribosomal frameshifting. To examine this possibility, a cDNA fragment containing the la-lb overlap region was cloned within a reporter gene and placed under the control of the bacteriophage SP6 promoter in a recombinant plasmid. Synthetic transcripts derived from this plasmid, when translated in the rabbit reticulocyte lysate cell-free system, specified the synthesis of polypeptides whose size and antibody reactivity were consistent with an efficient-1 ribosomal frameshift event at the overlap region. The HAst-l frameshift signal has two essential components, a heptanucleotide slippery sequence, A6C, and a stem-loop structure in the RNA. The presence of this structure was confirmed by complementary and compensatory mutation analysis and by direct structure probing with singleand double-stranded RNA-specific reagents. The HAst-l frameshift signal, like that present at the overlap of the gag and pro genes of the retrovirus human T-cell lymphotrophic virus type II, does not involve the formation of an RNA pseudoknot.

V, 2.Ribosomal frameshifting in astroviruses

“…An examination of the sequence information present within the overlap region of the two ORFs suggested that ORFlb was expressed as a translational fusion with the upstream la ORF following a -1 ribosomal frameshift event within the ORFla/lb overlap region (Jiang et al, 1993;Lewis et al, 1994;Willcocks et al 1994). This was subsequently confirmed experimentally in in vitro translation systems; the HAstV signal was shown to cause some 5-7% of ribosomes to frameshift (Marczinke et al, 1994;Lewis and Matsui, 1995). The frameshift signal of HAst-1 is one of the simplest described to date, comprising the slippery sequence AAAAAAC and a small GC-rich stem-loop located some six nucleotides (nts) downstream (see Fig.…”

“…HTLV-II gag/pro 5' (Marczinke et al, 1994), HTLV-II gag~pro (ten Dam et al, 1990), HIV-1 gag/pol (Jacks et al, 1988b;Kang, ] 998) and the pseudoknot of the coronavirus IBV (Brierley et al, 1991). The slippery sequences are underlined.…”

“…2). Frameshifting at this signal was tested by cloning a region of the astrovirus genome containing the putative frameshift signal into a plasmid-borne reporter gene and performing in vitro transcription and translation studies using the rabbit reticulocyte lysate (RRL) system (Marczinke et al, 1994). Site-specific mutagenesis confirmed the site of frameshifting as the AAAAAAC sequence, the presence of the stem region and also that the primary sequence of the loop nucleotides was unimportant, arguing against the presence of a pseudoknot.…”

“…Site-specific mutagenesis confirmed the site of frameshifting as the AAAAAAC sequence, the presence of the stem region and also that the primary sequence of the loop nucleotides was unimportant, arguing against the presence of a pseudoknot. Further experimental evidence that the HAst-1 stimulatory RNA is a stem-loop and not a pseudoknot was obtained by RNA structure mapping of the in vitro transcripts, which indicated that the loop nucleotides were accessible to single-strand specific chemical and enzymatic probes (Marczinke et al, 1994). A study of astrovirus frameshifting in an infection-transfection transient expression system also found that a minimal cassette of slippery sequence and downstream stem-loop was sufficient to induce efficient frameshifting both in vitro and in vivo arguing against the need for pseudoknot formation (Lewis and Matsui, 1996).…”

Stem-loop structure of Cocksfoot mottle virus RNA is indispensable for programmed −1 ribosomal frameshifting

“…Examples include the frameshift-promoting elements at the gag-pol junction in Giardia lamblia virus (GLV), Human immunodeficiency virus type 1 (HIV-1), Simian immunodeficiency virus (SIV) and related lentiviruses (Brierley and Dos Ramos, 2006;Li et al, 2001;Marcheschi et al, 2007;Staple and Butcher, 2005). The requirement for a simple hairpin loop in frameshifting has been also demonstrated in Human astrovirus serotype-1 (HAst-1), Human T-cell leukemia virus type II (HTLV-II) and Red clover necrotic mosaic virus RNA-1 (RCNMV) (Falk et al, 1993;Lommel, 1994, 1998;Marczinke et al, 1994).…”